Platelet reactivity expressed as a novel platelet reactivity score is associated with higher inflammatory state after coronary artery bypass grafting.

Introduction: Despite therapy, patients operated using a cardiopulmonary bypass demonstrate increased platelet aggregation, which rebounds to above preoperative levels. The aim of the study was to test the interaction between platelet reactivity/activation and selected inflammatory markers in the post-operative period. Material and methods: In total, 103 patients with non-ST elevation acute coronary syndrome (NSTE-ACS) who were not eligible for percutaneous coronary interventions (PCI), and required urgent revascularization, were included. Platelet reactivity was measured using the PFA-100 platelet analyser, multiple electrode aggregometry, and was expressed as a novel platelet reactivity score (PRS). Patients were divided using their PRS scores into high platelet relativity or low platelet reactivity subgroups (HPR or LPR). Platelet basal activation was measured using immunoassays for soluble P-selectin and soluble CD40L. We measured high-sensitivity C-reactive protein (CRP), and used immunoassays for tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) as inflammation markers. Results: Significant differences between HPR and LPR groups were found for CRP (mg/l): 81.5 vs. 44.6, p < 0.02; and TNF-α (pg/l): 3.51 vs. 2.37, p < 0.02. A significant association was found between CRP, TNF-α, IL-6 and platelet reactivity (platelet reactivity score). Cohen's k showed: CRP = 0.49, p < 0.0001, TNF-α = 0.37, p < 0.002. Perioperative myocardial infarction and rhythm disturbances occurred more frequently in the high platelet reactivity group: 7 (16.3%) vs. 2 (3.3%), p < 0.04, and 9 (20.9%) vs. 4 (6.7%), p < 0.04, respectively. Conclusions: Inflammatory parameters CRP and TNF-α are strongly associated with platelet reactivity (expressed as PRS) in cardiopulmonary bypass graft patients. Platelet hyperreactivity in the early post-operative period combined with a systemic inflammatory state correlates with a higher risk of post-operative rhythm disturbances and myocardial infarction.

Interactions of fentanyl with blood platelets and plasma proteins: platelet sensitivity to prasugrel metabolite is not affected by fentanyl under in vitro conditions.

BACKGROUND: Clinical trials indicate that fentanyl, like morphine, may impair intestinal absorption and thus decrease the efficacy of oral P2Y12 inhibitors, such as clopidogrel, ticagrelor, and prasugrel. However, the ability of fentanyl to directly negate or reduce the inhibitory effect of P2Y12 receptor antagonists on platelet function has not been established. A series of in vitro experiments was performed to investigate the ability of fentanyl to activate platelets, potentiate platelet response to ADP, and/or diminish platelet sensitivity to prasugrel metabolite (R-138727) in agonist-stimulated platelets. The selectivity and specificity of fentanyl toward major carrier proteins has been also studied. METHODS: Blood was obtained from healthy volunteers (19 women and 12 men; mean age 40 ± 13 years). Platelet function was measured in whole blood, platelet-rich plasma and in suspensions of isolated platelets by flow cytometry, impedance and optical aggregometry. Surface plasmon resonance and molecular docking were employed to determine the binding kinetics of fentanyl to human albumin, α1-acid glycoprotein, apolipoprotein A-1 and apolipoprotein B-100. RESULTS: When applied at therapeutic and supratherapeutic concentrations under various experimental conditions, fentanyl had no potential to stimulate platelet activation and aggregation, or potentiate platelet response to ADP, nor did it affect platelet susceptibility to prasugrel metabolite in ADP-stimulated platelets. In addition, fentanyl was found to interact with all the examined carrier proteins with dissociation constants in the order of 10-4 to 10-9 M. CONCLUSIONS: It does not seem that the delayed platelet responsiveness to oral P2Y12 inhibitors, such as prasugrel, in patients undergoing percutaneous coronary intervention, results from direct interactions between fentanyl and blood platelets. Apolipoproteins, similarly to albumin and α1-acid glycoprotein, appear to be important carriers of fentanyl in blood.

Systemic immune-inflammation index (SII) and neutrophil to lymphocyte ratio (NLR) are useful markers for assessing effects of anti-inflammatory diet in patients before coronary artery bypass grafting.

BACKGROUND: One of the risk factors responsible for coronary artery disease (CAD) is an inadequate diet that is frequently deficient in anti-inflammatory components, such as polyphenols and omega-3 fatty acids. The neutrophil to lymphocyte ratio (NLR) and the systemic immune-inflammation index (SII) are inflammatory markers that may reflect a diet's antiinflammatory potential. OBJECTIVE: The aim of this study was to evaluate the effects that CAD patients' nutrition patterns have on NLR and SII. MATERIAL AND METHODS: A retrospective study assessed the dietary habits and inflammatory marker levels in patients with advanced CAD before they underwent coronary artery bypass grafting (CABG) (n=101). Patients were divided into subgroups based on their NLR and SII levels. RESULTS: Subgroups with lower NLR and SII levels had consumed significantly more eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (p=0.02). The group with a lower ratio of omega-6 to omega-3 fatty acids (<4:1) also had lower NLR and SII levels (p=0.007 and p=0.01, respectively). Statistically significant negative correlations were found between EPA and DHA, as well as omega-3 intake, and both NLR and SII values. No statistically significant differences were found between the subgroups with lower and higher NLR and SII values for polyphenol intakes. CONCLUSIONS: Inflammatory markers such as NLR and SII may reflect an anti-inflammatory diet consumed by cardiac patients. A simultaneous assessment of dietary habits and inflammatory parameters is beneficial in the possible prevention of adverse cardiovascular incidents after CABG. There is also a need to establish reference values for SII and NLR.

Dietary intake of omega fatty acids and polyphenols and its relationship with the levels of inflammatory markers in men with chronic coronary syndrome after percutaneous coronary intervention.

BACKGROUND: The platelet­to­lymphocyte ratio (PLR), neutrophil­to­lymphocyte ratio (NLR), and C­reactive protein (CRP) are useful in assessing inflammation in patients after percutaneous coronary intervention (PCI). The PLR and NLR are also independent predictors of cardiovascular mortality. Moreover, higher CRP levels increase the risk of long­term mortality in patients undergoing PCI. AIMS: We aimed to investigate the relationship between the dietary intake of omega­3 and omega­6 fatty acids and plant polyphenols and the levels of inflammatory markers in patients after PCI. METHODS: In this retrospective study, we used the validated Food Frequency Questionnaire and Aliant software to estimate the dietary intake of polyphenols and omega­3 fatty acids as well as the ratio of omega­6 to omega­3 fatty acids in patients after PCI. A total of 105 patients were divided into subgroups based on high or low dietary polyphenol intake, omega­3 fatty acid intake, and omega­6 / omega­3 fatty acid ratio. Data on complete blood count were obtained from the hospital laboratory. RESULTS: In this retrospective study, we used the validated Food Frequency Questionnaire and Aliant software to estimate the dietary intake of polyphenols and omega­3 fatty acids as well as the ratio of omega­6 to omega­3 fatty acids in patients after PCI. A total of 105 patients were divided into subgroups based on high or low dietary polyphenol intake, omega­3 fatty acid intake, and omega­6 / omega­3 fatty acid ratio. Data on complete blood count were obtained from the hospital laboratory. CONCLUSIONS: Anti­inflammatory effects of a diet should be assessed not only based on a high intake of omega­3 fatty acids but also balanced omega­6 / omega­3 ratio, which reduces PLR and CRP levels in patients with cardiovascular disease.

Polyphenol-rich diet is associated with decreased level of inflammatory biomarkers in breast cancer patients

Background: The study investigated the relationship between dietary intake of polyphenols and inflammatory markers: CRP, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), medium platelet volume/lymphocyte ratio (MPVRL), in newly-diagnosed breast cancer patients. Objectives: The aim of this work was to verify whether diet rich in plant polyphenols affects inflammatory markers in breast cancer patients. Materials and methods: 78 patients (55.3±14.5 years) treated surgically for breast cancer were studied. A modified FFQ and authorial worksheet based on the Phenol Explorer database was used to measure the amount of plant polyphenols in a diet. Basing on the median of polyphenols intake (1780 mg/day), the group was divided into two subgroups: low- and high- dietary intake of polyphenols (LDIP and HDIP, respectively). Plasma CRP level was measured and NLR, PLR and MPVLR were calculated using results from peripheral blood morphology. Results: LDIP was associated with significantly higher CRP (elevated in 34.5% LDIP patients vs. 8.3% HDIP, p<0.003), NLR (elevated in 46.2% LDIP patients vs. 25.6% HDIP, p<0.006) and PLR level (elevated in 25.6% LDIP patients vs. 12.8% HDIP, p<0.03). MPVLR was not significantly different between both the subgroups. Conclusion: High dietary intake of polyphenols remarkably reduced process of inflammation in breast cancer patients, which has important clinical implications. The study demonstrated also an usefulness of simple, cheap and commonly available biomarkers for monitoring anti-inflammatory effects of diet.

Dual Anticoagulant/Antiplatelet Activity of Polyphenolic Grape Seeds Extract.

Because of the side-effects of commonly used anti-platelet and anticoagulant drugs, investigations into plant substances with similar activities are very common. Based on our own studies in recent years, we estimate that it is possible to use natural compounds to both inhibit coagulation pathway enzymes and to reduce blood platelets' activation. As such, in our current study we wanted to verify the anti-platelet and anticoagulant properties of grape seed extract (GSE) using in vitro models. During our analysis, the following parameters were analyzed: Coagulation times, thromboelastometry assays (coagulation time, clot formation time and maximum clot firmness), aggregation of platelets and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Adenosine diphosphate (ADP)-induced aggregation was lower in GSE 7.5 µg/mL as well as in GSE 15.0 µg/mL. A similar dependence was observed in VASP assays for GSE 7.5 µg/mL and GSE 15 µg/mL. The effect on plasma coagulation tests was distinct only with GSE 15 µg/mL. All of the thromboelastometry variables were statistically significant with 15.0 µg/mL GSE concentration. Our results show, for the first time, the multi-potential effect of grape seed extract on coagulation systems, and clearly suggest that grape seed extract could be considered a promising nutraceutical in the prevention of cardiovascular thrombotic events caused by different mechanisms.

Differentiated mitochondrial function in mouse 3T3 fibroblasts and human epithelial or endothelial cells in response to chemical exposure.

Mouse 3T3 fibroblasts are commonly used for in vitro toxicity testing; however, their sensitivity to stimuli is not well defined. To assess the sensitivity of the 3T3 cell line, the study compared the changes in mitochondrial membrane potential (MMP) occurring after exposure to eight chemicals known to demonstrate pro-apoptotic activity (glycerol, isopropanol, ethanol, paracetamol, propranolol, cobalt chloride, formaldehyde and atropine). Five cell lines were used as follows: mouse 3T3 fibroblasts, human epithelial cells (A549, Caco-2 and HepG2) and human endothelial cells (HMEC-1). Cell sensitivity was assessed based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The 3T3 fibroblasts had the highest AUOC values and were the most sensitive to the action of all the examined chemicals, with the exception of formaldehyde. Significant changes in MMP between the 3T3 cell line and other cells were observed after cell treatment with atropine (A549, Caco-2 or HMEC-1 cells vs 3T3 cells, P < 0.05), propranolol (A549 vs 3T3 cells, P < 0.01; HepG2 vs 3T3 cells, P < 0.05), cobalt chloride (A549 cells vs 3T3 cells, P < 0.01) or ethanol (HMEC-1 vs 3T3, P < 0.05). Formaldehyde appeared the most toxic compound for Caco-2 cells (Caco-2 vs 3T3 cells, P < 0.05). The surface areas (AUOC) calculated for each other chemical and obtained for HepG2, Caco-2, A549 and HMEC-1 did not differ significantly between cell lines. We postulate that mouse 3T3 fibroblasts demonstrate significantly higher relative sensitivity to many agents with toxic potential.

A new approach for the assessment of the toxicity of polyphenol-rich compounds with the use of high content screening analysis.

The toxicity of in vitro tested compounds is usually evaluated based on AC50 values calculated from dose-response curves. However, there is a large group of compounds for which a standard four-parametric sigmoid curve fitting may be inappropriate for estimating AC50. In the present study, 22 polyphenol-rich compounds were prioritized from the least to the most toxic based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The studied compounds were ranked across three key cell indicators (mitochondrial membrane potential, cell membrane integrity and nuclear size) in a panel of five cell lines (HepG2, Caco-2, A549, HMEC-1, and 3T3), using a high-content screening (HCS) assay. Regarding AUOC score values, naringin (negative control) was the least toxic phenolic compound. Aronox, spent hop extract and kale leaf extract had very low cytotoxicity with regard to mitochondrial membrane potential and cell membrane integrity, as well as nuclear morphology (nuclear area). Kaempferol (positive control) exerted strong cytotoxic effects on the mitochondrial and nuclear compartments. Extracts from buckthorn bark, walnut husk and hollyhock flower were highly cytotoxic with regard to the mitochondrion and cell membrane, but not the nucleus. We propose an alternative algorithm for the screening of a large number of agents and for identifying those with adverse cellular effects at an early stage of drug discovery, using high content screening analysis. This approach should be recommended for series of compounds producing a non-sigmoidal cell response, and for agents with unknown toxicity or mechanisms of action.

Xanthohumol from hop cones (Humulus lupulus L.) prevents ADP-induced platelet reactivity.

Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 µg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets' surface (47.6 ± 15.8 for 1.5 µg/ml XN, 44.6 ± 17.3% for 3 µg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 µg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.

Relationship between high on aspirin platelet reactivity and oxidative stress in coronary artery by-pass grafted patients.

The aim of the study was to assess the responsiveness of blood platelets to acetylsalicylic acid (ASA) in patients following coronary artery bypass grafting (CABG) surgery with relation to oxidative and antioxidative plasma status. The study included 37 patients treated with the CABG procedure. During the first 24 h after CABG patients were given 300 mg of ASA with the following dose of 150 mg daily. The blood was collected before the procedure and 10 days after. Whole blood platelet aggregation induced with arachidonic acid, collagen and adenosine diphosphate (ADP) was performed together with whole blood generation of thromboxane B2 (TxB2). Oxidative stress was measured before and 10 days after CABG with total oxidative plasma status (TOS) and total antioxidative status of the plasma (TAS). TOS/TAS index was calculated. We observed a significant increase in the TOS and TOS/TAS index and ADP-induced aggregation 10 days after CABG in comparison with its level before operation. There was a significant decrease in the arachidonic acid-induced aggregation and serum TxB2 level. Patients with ADP-induced and collagen-induced aggregation in the upper quartile had significantly higher TOS and TOS/TAS index before (ADP) and after the operation (ADP and collagen). There were 19 patients (51%) with high on aspirin platelet reactivity after CABG who had also higher TOS and TOS/TAS index and lower TAS value in comparison with aspirin responders. Despite ASA use, increased oxidative stress after CABG can overcome its antiplatelet effect and increase platelet activation through other pathways.

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells?

PURPOSE: Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. MATERIAL/METHODS: After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. RESULTS: Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 µg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 µg gallic acid equivalent/ml. CONCLUSIONS: The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients.

OBJECTIVES: Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro-inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers. METHODS: Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB(2), and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated. RESULTS: No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P=0.009; P=0.04; P=0.004, respectively). In both studied groups serum level of TxB(2) and platelet aggregation under AA stimulation were lower than before treatment (P<0.001). CONCLUSION: Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.

Anticoagulant effect of polyphenols-rich extracts from black chokeberry and grape seeds.

Blood coagulation consists of a series of zymogens that can be converted by limited proteolysis to active enzymes leading to the generation of thrombin. Fresh plasma and human thrombin was incubated with extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5; 5; 50 µg/ml). The in vitro experiments showed that both extracts prolonged clotting time and decreased the maximal velocity of fibrin polymerization in human plasma. Moreover thrombin incubation with both extracts results in the inhibition of amidolytic activity of this enzyme. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states.

Comparison of the VASP assay and platelet aggregometry in the evaluation of platelet P2Y12 receptor blockade.

INTRODUCTION: It has been shown that incomplete blockade of platelet reactivity is a risk factor for future ischemic events in patients with cardiovascular diseases. Despite these findings, there is yet no gold standard of platelet reactivity estimation. The 2 most commonly used methods in platelet testing are platelet aggregometry and vasodilator-stimulated phosphoprotein phosphorylation (VASP) assay. They both showed the predictive value for future adverse events in cardiac patients; however, there are few data that compare these 2 methods. OBJECTIVES: The aim of the study was to compare the results of aggregometry (multi-electrode aggregometer [MEA]) and flow cytometric VASP assay used to determine platelet reactivity after the administration of P2Y12 receptor blockers. PATIENTS AND METHODS: The study included 17 healthy volunteers (12 men, 5 women; aged 41 ±10 years) and 12 patients (men, aged 62 ±12 years) with stable coronary artery disease treated with elective percutaneous coronary intervention with stent implantation. In volunteers, the blood was collected and tests were performed before and after 10-minute incubation with 5 nmol/l of cangrelol. In patients, the blood was collected for measurements before and after ingestion of 300 mg of clopidogrel. Aggregometry measurements included adenosine-diphosphate (ADP)-induced maximal aggregation (A(max)) and ADP-induced area under the aggregation curve (AUC). The platelet reactivity index (PRI) was determined using the VASP assay. RESULTS: The use of cangrelor and clopidogrel was associated with a significant inhibition of platelet reactivity measured using the above methods. In both groups, the degree of inhibition was significantly greater when measured with the aggregation method compared with the VASP assay. The only significant coefficient of correlation between the VASP assay and aggregation results was observed in volunteers after platelet incubation with cangrelor (r= 0.81 between PRI and A(max), r = 0.68 between PRI and AUC). CONCLUSIONS: Compared with the VASP assay, ADP-induced platelet aggregation shows a greater ability to detect a decrease in platelet aggregation after P2Y12 antagonists. These tests are not interchangeable because they measure different aspects of the P2Y12 receptor blockade.

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells.

INTRODUCTION: Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. MATERIAL AND METHODS: THIS STUDY ENCOMPASSES TWO MODELS OF MONITORING PLATELET REACTIVITY: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. RESULTS: We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. CONCLUSIONS: It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases.

Can we use adenosine diphosphate (ADP) to study "aspirin resistance"? The Janus faces of ADP-triggered platelet aggregation.

There is a growing number of contradictory reports indicating that adenosine diphosphate (ADP) can be a useful agonist in monitoring of the antiplatelet action of acetylsalicylic acid (ASA) in humans and animals. In the current study, we aimed to determine the conditions for using ADP to trigger platelet aggregation in order to detect ASA-mediated inhibition of rat platelet reactivity. Initially, we examined the usefulness of different ADP concentrations (0.25, 0.5, 1, 5 and 10 microM) in detecting the in vitro ASA mediated platelet inhibition using whole blood aggregometry, as well as we monitored the role of ADP in generation of thromboxane A(2) (TXA(2)). To study ex vivo ASA inhibitory potential on platelet aggregation induced by a range of ADP concentrations, animals were subjected to one or 10-day ASA administration at the dose of 50 mg/kg. Our experiment shows that ADP in a concentration-dependent manner induces TXA(2) generation in the whole blood with hirudin as an anticoagulant. However, in vitro and ex vivo examination of ASA inhibitory potential on platelet aggregation revealed that irrespectively of administration regimen, ASA failed to block platelet aggregation induced by ADP at the concentrations higher than 0.5 microM. Our findings suggest that the mechanism of ADP-induced platelet aggregation depends on agonist concentration. It appears that only low ADP concentrations (up to 0.5 microM) induce TXA(2)-dependent rat platelet aggregation. Therefore, ADP could be considered a useful platelet agonist for monitoring of ASA-mediated platelet inhibition only if used at much lower concentrations than those commonly employed.

Does pycnogenol intensify the efficacy of acetylsalicylic acid in the inhibition of platelet function? In vitro experience.

INTRODUCTION: Some compounds of herbal origin, such as Pycnogenol (PYC), have been considered as an aid in antiplatelet therapy. Pycnogenol, a French maritime pine bark extract, is a complex mixture of polyphenols that has the ability to reduce human smoking-induced platelet aggregation. The aim of this study was to evaluate the in vitro ability of PYC to improve the efficacy of acetylsalicylic acid (ASA) in the inhibition of platelet function. MATERIAL/METHODS: Whole blood, anticoagulated with hirudin, was drawn from 38 volunteers (40.4+/-13.8 years old) and incubated with PYC (10, 50, 100 microg/ml) or/and ASA (25, 100 micromol/l) for 20 min at RT.PYC was dissolved in water (water-PYC group, n=20) or ethanol (ethanol-PYC group, n=18). To investigate platelet functions, PFA-100 closure-time determination, whole-blood electrical aggregation (WBEA), and PRP aggregation were employed. Collagen (1 microg/ml) and ADP (5 micromol/l) were used as platelet agonists. RESULTS: A compounding effect of ASA and PYC to inhibit platelet function recorded in collagen-induced aggregation in PRP was observed, but only when ethanol-dissolved PYC was used. The inhibitory effect of PYC (alone) was most profound in platelets activated with ADP. At all concentrations, PYC significantly inhibited platelet aggregation only in the ethanol-PYC group. CONCLUSIONS: It was found that under in vitro conditions, ethanol-dissolved PYC deepened the efficacy of ASA to inhibit platelet function. This study confirmed the direct and compounding (with ASA) inhibitory effect of PYC on platelets. These observations encourage the concept that the combined use of ASA and PYC may be beneficial in patients with impaired response to ASA therapy.

Usefulness of whole blood aggregometry and its comparison with thromboxane generation assay in monitoring acetylsalicylic acid effectiveness--a multiparametric study in rats.

BACKGROUND: There is a need for consensus concerning universal methodological criteria for detection of suboptimal response to acetylsalicylic acid (ASA) therapy. Therefore, animal models to test for ASA effectiveness remain of interest. Our objective was to verify the usefulness of multiparametric whole-blood impedance aggregometry and thromboxane A(2) generation, which are the most popular techniques used for monitoring of ASA treatment effectiveness. METHODS: Using multiparametric analysis of whole-blood impedance aggregometry, we examined which parameters of platelet aggregation or disaggregation allow for the best discrimination between ASA-treated (4 or 40 mg/kg for 60 days) and non-treated male rats. The effectiveness of ASA-mediated inhibition of platelet cyclooxygenase-1 was verified by determination of plasma thromboxane B(2) and urine 11-dehydro-thromboxane B(2), accepted as reference assays for monitoring of ASA-mediated platelet cyclooxygenase-1 inhibition. RESULTS: Two of the platelet agonists used, collagen (1 mg/L) and arachidonic acid (0.5 mmol/L), allowed discrimination of control and ASA-treated animals, whereas adenosine diphosphate (5 micromol/L) was not effective. It is noteworthy that only ASA-mediated changes in duration of the rising phase for platelet aggregation and the area under the curve for collagen-induced aggregation allowed significant discrimination between low and high ASA dose and remained correlated with the reference parameter, plasma thromboxane B(2). CONCLUSIONS: Analysis of aggregation curves, routinely based only on the amplitude and rate of platelet aggregation, may not be enough discriminative to distinguish between varying ASA doses and treatment schedules.

Resistance to aspirin in patients after coronary artery bypass grafting is transient: impact on the monitoring of aspirin antiplatelet therapy.

The aim of the present study was to assess the responsiveness of blood platelets to aspirin in patients following coronary artery bypass grafting (CABG) surgery. Aspirin was administered following CABG in 24 operated patients (aged 63.2 +/- 6.3 years). Platelet function was monitored on the 10th postoperative day (A) and 1 month after CABG (B) with the use of whole-blood aggregometry (WBEA) and PFA-100 closure time (PFA-100 CTCEPI). Normal platelet response to aspirin was defined by 3 criteria: the complete inhibition of WBEA induced by arachidonic acid (0.5 mmol/L), partial inhibition of collagen (1 microg/mL)-induced aggregation (WBEA < 14 Omega), and prolongation of PFA-100 CTCEPI (>150 seconds) ("good responders"). Depending on whether 0, 1, 2, or 3 of these 3 criteria were fulfilled, patients were classified as "nonresponders," "weak responders," "incomplete responders," or "good responders," respectively. On the 10th postoperative day, there were 3 good responders, 6 incomplete responders, 11 weak responders, and 4 nonresponders among 24 patients. In contrast, 1 month after CABG within the same group of 24 patients there were 18 good responders, 5 incomplete responders, and 1 weak responder. Using a new methodology to assess impaired platelet responsiveness to aspirin ex vivo, we describe here the transient nature of "aspirin resistance" following CABG. These results indicate the necessity for the prolonged monitoring of the antiplatelet effectiveness of aspirin in the postoperative period after CABG.

Limited usefulness of the PFA-100 for the monitoring of ADP receptor antagonists--in vitro experience.

We have evaluated the usefulness of the PFA-100 system (collagen/ADP and collagen/epinephrine cartridges) to assess the in vitro effects of a few platelet function inhibitors: Aspisol (60 microg/ml), 4-[4-[4-(aminoiminomethyl]-1-piperazinyl]-1-piperidineactetic acid, hydrochloride trihydrate (GR144053F, fibrinogen receptor antagonist, 100 nM), adenosine-3',5'-diphosphate (A3P5P, P2Y1 ADP receptor antagonist, 500 microM) and Bis[(adenosine-5'-O-phosphorodithioyl)methylene]-phosphinic acid (APTMPA, P2Y12 ADP receptor antagonist, 500 microM) on platelet function, as compared with the other commonly used diagnostic technique, a whole blood electrical aggregometry (20 microM ADP or 0.5 mM arachidonic acid). The in vitro studies were carried out on a group of 38 subjects. Whereas all the examined platelet antagonists and inhibitors almost completely blocked the 20 microM ADP- or 0.5 mM arachidonic acid-induced (in the case of acetylsalicylic acid) whole blood aggregation, only two inhibitors (Aspisol and GR144053F) remained effective in a significant prolongation of the PFA-100 occlusion time. Otherwise, using the PFA-100 system we were not able to detect the inhibitory actions of ADP receptor antagonists- P2Y1 and P2Y12. Our findings point to a limited usefulness of the PFA-100 system for the monitoring of the effectiveness of ADP receptor antagonists. The outcomes of this study show that platelet aggregometry in whole blood is characterised by the highest sensitivity in the monitoring of the investigated blood platelet inhibitors.